Nutrient media containing antifoaming agents



Patented Jan. 12, 1954 httddi? NUTRIENT MEDIA CONTAINING ANTIFOAIYIING AGENTS Henry V. Moss and Roy E. Morse, Anniston, Ala., assignors to Monsanto Chemical Company, St. Louis, Mo., a corporation of Delaware 1 No Drawing. Application July 14, 1950, Serial No. 173,936

10 Claims.

The present invention relates to a method of inhibiting, diminishing or preventing the development of excessive amounts of foam in the manufacture of yeast, molds, bacteria, related microorganisms of fermentation, their derivatives and by-products such as penicillin, streptomycin, etc. It also relates to substantially non-foaming nutrient media which are eminently suitable for use in the production of the above products, and to the method of preparing same.

in accordance with current practice in the fermentation industry, yeasts, molds, bacteria, related microorganisms of fermentation, their derivatives and Icy-products such as penicillin, streptomycin, etc. are propagated or produced in suitable nutrient media containing carbohydrates such as sugar, starch, etc., a usable source of nitrogen, nutrilites and various salts which supply essential mineral elements. These nutrient media are divided into two general classes, namely, natural and synthetic.

Natural nutrient media are the juices of fruits or plants in their natural state. For example, the expressed juices of grapes, apples, pears and other fruits are excellent media since they contain sugars along with nitrogen-containing compounds and dissolved salts at a pH favorable to the development of the above microorganisms of fermentation, their derivatives and by-products.

Synthetic media are artiiically prepared aqueous compositions including sugars and various inorganic salts and organic compounds. A typical example is one prepared by mixing together in suitable proportions crude beet molasses, calcium phosphate, ammonium monohydrogen phosphate, urea, concentrated sulfuric or phosphoric acid and water.

In the commercial production of microorganisms of fermentation, their derivatives and by-produots, it has been found essential to aerate the nutrient media during propagation in order to produce the desired roduct in good yields and in a minimum amount of time. For example, if aeration is not employed in the manufacture of yeast. the propagation of this product proceeds at a relatively slow rate. Moreover, the yield of alcohol is increased at the expense of the yeast with the result that the yield of the latter is substantially reduced. This highly undesirable result is overcome by the above expedient, but it introduces a serious foaming problem which has been attacked by mechanical and/or chemical clefoaming means. However, for one reason or another they have not been entirely satisfactory, and, consequently, there has been an urgent demand by the fermentation industry for a solution to this problem.

It is, therefore, an object of the present invention to provide a commercially and economically feasible method and means of inhibiting, diminishing or preventing the development of excessive amounts of foam in the manufacture of yeast, molds, bacteria, related microorganisms of fermentation, their derivatives and lay-products.

Another object of the invention is to provide substantially non-foaming nutrient media which are admirably suitable for use in the commercial manufacture of the above products.

A further object of the invention is to provide a new series of antifoaming agents for use in the fermentation industry.

Additional objects and advantages will be apparent to those skilled in the art as the description of the instant invention proceeds.

In accordance with the present invention, the surprising discovery has been made that if a relatively small amount of an ethylene oxide derivative of the type hereinafter described is added to natural and/or synthetic nutrient media used in the propagation or production of microorganisms of fermentation, their derivatives and by-products, excessive foaming of the resulting media, upon aeration, is effectively inhibited or minimizecl. Stated in a different manner, the media treated in the above manner are characterized by the fact that they are substantially non-foaming or are stabilized against the development of excessive amounts of foam upon aeration.

The amount of ethylene oxide derivative used to inhibit or minimize foaming may fluctuate within wide limits depending upon various conditions hereinafter described, but it is desirable for economic reasons not to use more than 10 by weight of the above material. In general, about 0.001% to about 3% and preferably about 0.1% to about 0.2% by weight of the eth lene oxide derivative is sufiicient to solve the foaming problem encountered in most situations in the ermentation industry. However, it is to be understood that the invention is not restricted to the above numerical limits since larger or smaller amounts may be used if required.

The ethylene oxide derivatives contemplated by the instant invention are those prepared by condensing abietic acid or products containing same such as rosin, rosin oil or tall oil with about 0.75 to about 6 moles of ethylene oxide per mole of abietic acid or tall oil. Stated in terms of parts by weight, the above derivatives are prepared by condensing about 1 to about 9.5 parts by weight of rosin or rosin oil (basis abietic acid) with 1 part by weight of ethylene oxide or by condensing about 1 to about 10.5 parts by weight of tall oil (average molecular weight: 290-338) with 1 part was allowed to trickle upwardly through the fritted disc to produce small air bubbles.

The foam producing material employed in the above apparatus was prepared in the following by weight of ethylene oxide. 5 manner.

By mole oftall oil ismeant the'averagemolecu- A commercial cane syrup wasadjusterl to a pH lar weight of the components oftall-oil of 4.5-by theaddition of hydrochloricacid,"where- The method of preparing ne of the ab upon it was diluted with water to a specific gravethylene oxide derivatives is described in detail in y of 1-039 at Q/200 ThiS P' Was application, 3. N. 122,585, filed October.20, 1.959, hen ,packed into pin h canning j p in the name of Earl W. Gluesenkamp, which .apfiervefiihymrocessing in ili W r nd co led plication is assigned to the same assignee as the y means of Using this y p, e foa present invention and is incorporated by re'ferliolming material P ced by miXi g the ence into the present specification. The method Fformehwithibfihersyeast in the DTODOTtiOTIS e of preparing the other derivatives is thesame exina'fter indicated and then mechanically stirring cept for the variation in ratio of ethylene oxide Lthemixtureior Eminutes to insure p e 5 to tall oil or abietic acid. zpenSiOh 0 e y ast- Tall oil is subject to considerable variation in In practice, the bath and foam towel were composition being in part dependent on the brought -to-a temperature of about 30 C. and the cies of ood proceqsed the ea on of the introduction Of air initiated. Then, the abovethe tree va .cut zthe.,a;ge.of th-eitree the sto ag described. foam-forming material VIBJS added to life of the wood. the rocedure for processing the the ioam tower continuously aerated to chips'and converting theblack'liduor into-tall oil, activate thelyeast- The reactivation of thelyeast eta Thus the :average analysis of 10931 on from could be recognized readily by the character of fifteen Wins ranged vto 54 nementfattv the resulting foam since it was quite different id -381- 5g Rampant rosin amiss and to from that produced prior to reactivation; 7 this per ent un aponifiable. In view of th abo difference was probably due to cell surface variat on in-cnmoosition. it can be seen that the changes brought? change'ofer from avera e molecular weight of tall "oil will vary qmescence to aCtIYG'YQSQI1atiOnacmrdingly As soon as the yeast had been reactivated'and For 2' more. Coma-16m nnaewtqnmnmdflhp nves the'foamhad begun to rise in the tower, the antient invpnflon.rpferenne-is mgfie tn flqQ-fnunvfing foaming agent was added and the time of the descfintinn of mp-fhofi and .annamtnsmspfl .3130 addition'observed. Thereafter; the time required evaluate. the ant foarnine nmp'erties 'nfith' Joli-{hob for failure of the antifoaming agent was observed ene oxide derivatives .megent mWfih-nn 3'5 and recorded. After failure of the antifoaming and also to the ex erimental data he e nafter agent the yeast Separated from the presented, which illustrate the gefiectivenpss of washed three times withcolddistilled water and the above compounds-in :nreventingor inhibiting ()bseWediq'Yiabmty and'odorthe fievelonment of excessiveioam: The viability of the yeast was determined .by

The avpmmtus [consisted egsentiquvvof meow 40 its appearance and'behavior on the filter. Since stant temperature water bath eoluin ed with a the tests had been run for less stirrer. a heating coil and suitable thermostatic complete exhaustion Syrup by the yeast controls by which the bath temperature was was no.1; achleied therefore gas evohttion maintained at 30 C.: :0I2 C.: a tubular pyrex W FP P the'filter pad due to l f foam tower (48 mm. in diameter "and 55cm. in g z g on i q lfwhe yeast length) standing in the above bath said tower a l een damaged e antlfoammg'agent respiratory gases were apparent on the filter pad, 8! P Q at a l of 3 mfrom 1135 and, moreover, the cell :morphology was altered f 1th a coarse {fitted also; of with the result that small,.closeepacking cellsand Whlch was Conducted through a glass wool 50 cellularcontents wereformed whichmarkedl-y infilter, an orifice flow meter and .a glass take-off creased th fi1t rj n time tube, passing downwardly through the bath into Using t abovedescribed methcd f ,1 1; the tower ata point immediately aboveitsbottom. ing antifoamingagents, tall oil and various ethyl- :Ihe air was conveyed .at the rate .of 0.125 cu. ene-oride derivatives thereof were tested with the ft./min. into the bottom of the tower where results indicated in the following table.

Table 0 i'of Toxisfity of A in Composition'ofnntrienhmedmm Antifoam'mg gen filgghed figgz fit i r i ti ni e t e r yeast :ouyeast hours }Rc(jfin ejdb%%l)l oil (59. gr. at 25 0.125" Normal- Non toxic. 2.5 15.0. Antifoaming agent, 0.05 ml... Cane syrup, 104.0 gms Condensation product of ethylene do "don"; 0.5 45, Bakers yeast, 1.0 gms oxide with refined tall oil inmolar Antifoaming agent, 0.2 m1 ratio of 0.25:1. I V Cane syrup, 104.0 gms Condensation product of ethylene do do 05 40. Bakers yeast 1.0 gms. i oxide with refined tall oilin molar Antifoaming agent, 0.2 m1. ratio of 0.50 1. Cane syrup, 104.0 gms Condensation product of ethylene .'do "do"... -6.-0- :10. Bakers yeast, 1.0 gins oxide with refined tall 011 in molar Antifoammg agento'z m1 cgiiigrigagigi mduot of ethylene do do 4 0 0-5 gg gg iW g- 2078 oxide with refined tall oil igl molag h I inwalisglant,,0.osiai:;:;:;:...::i gf of a it 25 Table-Continued Toxicity of Odor of Aeration Composition of nutrient medium Antiioaming agent finished fi gzg time in ig gfi ifigg yeast on hours yeast Condensation product of ethylene Normal. N on-toxic. 7.0 0.5. gg fig igg fi h oxide with refined tall oil molar Antifoaming agent, 0.21111 at 25 CR5 Cane syrup (sp. gr. 1.039), 207.8 gnis Condensation product of ethylene do do... 6.5 0.5. Bakers yeast, 2.0 glllS oxide with refined tall oil in molar Antifoaming agent, 0.05 ml Cratiio of 2551. d t I th 1 d d 6 0 on ensa ion pro no 0 e y ene o on..- ggi g iygg g 5 207's oxide wigigrrifined tall gllzn nogg r. a Amfioammg agent, M5 1111 cgggbout 102 5); i I th d d 0 0 5 on ensation pro uc o e y ene o o 6. ggflg glgg g g 62 15 207's oxide with refined tall oil in molar Antitoaming agent, otter-I311: {E 3 at 1/25 Cane syrup, 52.0 gms Condensation product of ethylene do .do 18 8 Bakers yeast, 0.5 gm oxide with refined tall oil In molar Antifoamlng agent, 0.2 ml ratio of 6:1. Cane syrup, 52.0 gms Condensation product of ethylene do do. In excess of 55. Bakers yeast, 0.5 gm oxide with refined tall oil in molar Antiioaming agent, 0.2 ml Cratio of 1:1. d t f t 1 d d 0 1 200 on ensa ion pro us 0 e yene o o restaurateur}:fries: with an 5 5,2 eggs Antifoarning agent, 0.05 ml 8?: B 8: a

The above table demonstrates that tall oilethylene oxide condensation products containing about 0.75 mole to about 6 moles of ethylene oxide per mole of tall oil have outstanding antifoaming properties, whereas tall oil and tall oil-ethylene oxide condensation products containing less than 0.75 mole of ethylene oxide or more than 6 moles of ethylene oxide per mole of tall oil are of no value as they either induced foaming or had no antifoaming action at all.

The foregoing experimental data have been restricted to nutrient media for yeast propagation, but it is to be understood that the invention is not limited thereto. On the contrary, as indicated earlier in this specification, the invention is of much wider scope being broadly applicable to the solution of the foaming problem encountered in nutrient media used in the propagation of microorganisms of fermentation generally. In this connection, the following are cited as illustrative examples of substantially non-foaming nutrient media which are within the scope of the instant invention.

Nutrient media for yeast and molds Grams Condensation product of ethylene oxide with tall oil inmolar ratio o1'1:1 0. 25 0. 25 0. 25 Water 100.00 100. 00 100.00

Nutrient medium for penicillin Percent by weight Lactose 2.500 Cerelose .500 Corn steep liquor solids 2.000 ZIISO4 0.005 CaCO3 0.100 Precursor 1 0.050

hyde, phenylacetic acid, and phenylacetylglycine.

Nutrient medium for penicillinContinued Percent by weight Vegetative inoculum (Penicillium notatum) 7.000 Condensation product of ethylene oxide with tall oil in molar ratio of 1 3 1 3.000 Water 34.815

Nutrient medium for streptomycin Percent by weight Carbohydrate (dry weight) As pointed out earlier herein, it is not desirable for economic reasons to add more than 10% by weight of the ethylene oxide derivative to nutrient media used in the propagation of yeast, molds, bacteria, related microorganisms of fermentation, their derivatives and by-products such as penicillin, streptomycin, etc., but more may be used if required. In general, about 0.001% to about 3% and preferably about 0.1% to about 1.0% by weight of the antifoaming agent is sufiicient to overcome the foaming encountered in the use of the above media.

The minimum, optimum and maximum amounts of ethylene oxide derivative which may be used to inhibit foaming depends upon one or more of a number of conditions such as the selected antifoaming agent; the microorganism of fermentation to be propagated; the composition, pH and temperature of the nutrient medium; the volume to surface ratio of the nutrient medium; the bubble size; the air pressure used in aerating; and the rate of aeration and aeration time. Therefore, any attempt to numerically define the concentration of the antifoaming agent so as to meet all situations would be impractical and meaningless. However, since this may be readily determined by one skilled in the art, suflice it to say that it may be generically defined by stating that the antifoaming agent is employed in an amount suflicient to substantially inhibit foaming.

Although the present invention has been described with reference to a particular embodiment, it will be apparent to those skilled in the art that variations and modifications thereof can be made without departing from the principles and true spirit of the invention.

We claim:

1. An aqueous nutrient fermentation medium normally having a tendency to foam excessively and a sufficient amount'of an antifoaming agent to render said medium substantially non-foaming, said agent being the condensation product of about 0.75 to about Smols of ethylene oxide with 1 mole of a material selected from the group consisting of abietic acid and products containing same.

2. An aqueous nutrient fermentation medium normally having a tendency to foam excessively and about 0.001% to about by weight of an antifoaming agent, said agent being the condensation product of about 0.75 to about 6 moles of ethylene oxide with 1 mole of a material selected from the group consisting of abietic acid and tall 011. I

3. An aqueous nutrient fermentation medium normally having a tendency to foam excessively and-about 0.001% to about 3% by weight of an antifoaming agent, said agent being the condensation product of from 1 to'6 moles of ethylene oxidewith 1 mole of-a material-selected from the group consisting of abietic acidand tall oil.

4. An aqueous nutrient fermentation medium normally having a' tendency to foam excessively and about 0.1% to about 0.2% by Weight of an antifoaming agent, said agent being the condensation product of 1 mole of ethylene oxide with 1 mole of a material selected from the group consisting of abietic acid and tall oil.

5. An aqueous nutrient fermentation medium normally having a tendency to foam excessively and about 0.1% to about 0.2% .by weight of an antifoaming agent, said agent being the --condensation product of 1 mole of ethylene oxide with 1 mole of tall oil.

.6. The .method of substantially inhibiting the development of foam in aqueous nutrient fer-mentation :media normally having a tendency :to Jioam excessively, whichcomprises adding to said media a sufficient amount of .an antifoaming agent to inhibit foaming, said agent being :the condensation :product of about 0.75 to about -6 moles of ethylene-oxide with 1 mole'of a material selected from :the group consisting of :abietic acid and products containing same.

7. The method of substantially inhibiting the .developmentof foam in aqueous nutrient fermentation media normally having a tendency to foam excessively, which comprisesadding .to said media about 0.001% to about 10% by weight of an antifoaming agent, said .agent being the condensation productof about 0.75 to about 6 moles of ethylene oxide with 1 mole of a material selected from the group consisting .of abietic acid and tall oil.

8.The method of substantially inhibiting the development of foam in aqueous nutrient fermentation media normally having a tendency to foam excessively, which comprises adding to said media about 0;001% -to about 3% by weight of an antifoaming agent, said agent being the condensation-productof from 1 to 6 moles of ethylene oxide with 1 =moleof a material selected from the group consisting of abietic acid and .tall oil.

v9. The method of substantially inhibiting the development of foamin aqueous nutrient fermentation media normally having a tendency to foam excessively, which comprises adding to said media about 0.1% to about 0.2% by weight of an antifoaming agent, said agent being the condensation product .of 1 .mole of ethylene oxide with .1 mole of .amaterial selected from the group consisting of abietic acid and tall oil.

10. The .method of substantially inhibiting the development of foam in aqueous nutrient fermentationmedia normally having a tendency to foam excessively, which comprises adding to said media about 0.1% to about 0.2% by weight of an antifoaming agent, said agent being the condensation product of 1 mole of ethylene oxide with .1 mole of tall oil.

HENRY V. MOSS. ROY E. -MORSE.

References Cited in the file of this patent UNITED STATES PATENTS Number :Name Date 2,443,989 'Moyer June 22, 19. 28 2,448,790 Foster etal. Septfll, 1943 2,469,493 Barker "May 10, 1949 2,523,245 .iCoppock :Sept. 19, 195.0 2,550,450 vliiroiwlnietial Apr. 24, 1951 FOREIGN PATENTS Number Country Date 467,571 Great Britain June 16, 1937 OTHER REFERENCES Bennett, Monoand di-glycerides, Food Manufacture, July 5, 1940,'page 188. 

1. AN AQUEOUS NUTRIENT FERMENTATION MEDIUM NORMALLY HAVING A TENDENCY TO FOAM EXCESSIVELY AND A SUFFICIENT AMOUNT OF AN ANTIFOAMING AGENT TO RENDER SAID MEDIUM SUBSTANTIALLY NON-FOAMING SAID AGENT BEING THE CONDENSATION PRODUCT OF ABOUT 0.75 TO ABOUT 6 MOLS OF ETHYLENE OXIDE WITH 1 MOLE OF MATERIAL SELECTED FROM THE GROUP CONSISTING OF ABIETIC ACID AND PRODUCTS CONTAINING SAME. 